PROJECT SUMMARY Beginning as a simple tube of endoderm surrounded by mesenchyme, the gut is patterned early in development along its radial axis to generate discrete layers of smooth muscle with distinct orientations. Correct organization of these muscle layers into circumferential and longitudinal bands is required for peristalsis and to generate appropriate physical forces that drive folding of the gut lumen. Yet, how the radial pattern of muscle is determined with precise spatiotemporal resolution during development is not known. Building upon work from our lab and others, we predict that this precision is achieved through specific levels of Hedgehog (Hh) and BMP signaling activity within the gut mesenchyme. By modulating levels of Hh pathway activity, we show in preliminary studies that both low and high levels of Hh signaling inhibit differentiation of the early-forming circumferential smooth muscle layer. We predict that this inhibitory effect of Hh at high levels is due to increased Hh-dependent BMP signaling activity, which is activated at high threshold levels of Hh and acts to block muscle differentiation. Importantly, when Hh signaling is only slightly decreased smooth muscle smooth muscle forms adjacent to the endoderm. Given these data, we put forth and test model in Aim 1 in which the circumferential band of smooth muscle forms at an exact radial location where Hh signaling is sufficiently high, yet the inhibitory effects of BMP are low enough to not interfere. What then triggers the formation of the outer and inner longitudinal layers that form subsequent to the circumferential layer? Our preliminary results indicate that continuous BMP signaling acts to suppress the differentiation of the longitudinal layers, as exogenous Noggin causes their precocious differentiation. We additionally show that Noggin is expressed specifically by differentiating enteric neurons, and put forth a model wherein temporally specific and expression of Noggin in enteric neurons may serve to initiate the differentiation of longitudinal muscle at the correct place and time by locally antagonizing BMPs. In Aim 2, we test this model through embryological manipulations in chick and genetic loss of function in mouse to determine if Noggin from the NCCs is required for longitudinal muscle differentiation. Furthermore, we aim to identify the cellular origin of the longitudinal muscle. The results of the experiments proposed herein will establish a mechanistic model to explain smooth muscle patterning in the vertebrate gut and have the potential to improve our understanding of the mechanism underlying muscle dysplasias that impair digestion.